We propose to develope a set of powerful and versatile methodologies which will greatly expand the capability to successfully solve the most challenging classes of macromolecule crystal structures (membrane proteins, macromolecular complexes and functional RNAs). Our approach, Chaperone Assisted Crystallography (CAC), is based on utilizing "crystallization chaperones"-engineered binding domains targeted to bind to a chosen molecular entity- to facilitate crystallization and provide crystallographic phasing information. This approach has been proven to be spectacularly successful in crystallizing a number of important membrane proteins, where antibody fragments play a dominant role in forming effective lattice contacts. These successes suggest that the CAC strategy may be broadly applicable to a wide variety of structural biology targets, but they relied on the classic hybridoma approach for antibody production, which is cumbersome, nherently inefficient and expensive. To circumvent these technical problems, we will employ a powerful combination of novel antibody combinatorial libraries, and phage and yeast display technologies to produce the chaperone molecules. Our preliminary results show that a novel antibody combinatorial library, comprising a "reduced genetic code"; i.e., only four amino acid types, is as powerful in producing binding laffinity to a target, as one that contains all 20 amino acid types. The importance of this major breakthrough in protein engineering is that it allows a significant increase in chaperone sites that can be combinatorially diversified, and it is this feature that efficiently produces high affinity chaperones. We will use three chaperone classes based on different molecular scaffolds: an antibody Fab domain (approximately 400 amino acids), a camelid VHH domain (approximately 130 aa), and a fibronectin type III domain (approximately 90 aa). We will optimize reduced genetic codes for different scaffolds and different target classes. We will test the novel concept of "chaperone assisted lattice initiators," where we will engineer higher order chaperone assemblies for efficient crystallization. We will organize distinct technology modules of our approach in a high throughput pipeline format. Our technology is amenable to parallelization, and much of it can be disseminated to a small lab environment, providing powerful tools to attack major structure biology problems to the individual research groups. The chaperones produced in this project themselves will also be highly valuable tools in biomedical research and could supercede the hybridoma-produced antibodies.